Effects of leucism on organ development and molecular mechanisms in Northern snakehead (Channa argus) beyond pigmentation alterations

Leucism, a widespread occurrence observed in Northern snakehead (Channa argus), bestows a striking white jade-like body coloration upon affected individuals and has gained substantial popularity in commercial breeding. While the visible manifestation of leucism in snakeheads is primarily limited to body coloration, it is crucial to explore the potential influence of leucism on organ development and elucidate the underlying molecular mechanisms. Through a comparative analysis of growth differences, our study revealed that at 150 days post-fertilization, the white variety exhibited an 8.5% higher liver index and intestinal index, but experienced a 20% and 38% decreased in spleen index and renal interstitial index, respectively, suggesting an enlarged digestive area but relatively smaller immune tissues. Nonetheless, no significant differences were observed in the intestinal flora between the two varieties, suggesting the exclusion of any exogenous impacts from symbiotic flora on the growth and development of the white variety. Importantly, transcriptome analysis demonstrated that the white variety exhibited higher expression levels of innate immune genes. Furthermore, annotation of the gene sets expressed in the liver and spleen revealed 76 and 35 genes respectively, with the white variety displaying lower expression in genes associated with “Viral protein interaction with cytokine and cytokine receptor”, “Protein processing in endoplasmic reticulum”, and “TNF signaling pathway”, while exhibiting higher expression in “Estrogen signaling pathway”. Notably, three genes, namely pcdhf 4, nlrc3 card 15-like, and a pol-like were identified in both the liver and spleen, indicating their potential involvement in altering the development and innate immunity of the white variety. This study reveals the systemic impact of leucism that extends beyond mere pigmentation alterations, highlighting the prominent characteristics of this phenotype and providing a foundation for future molecular breeding programs aimed at enhancing this variety.

The Northern snakehead (Channa argus) belongs to the Perciformes order, Anabantoidei suborder, Channidae family, and the genus Channa.It is widely distributed in major water systems throughout China, characterized by its dark black body with irregular cloud-like black patches on each side 1, 2 .C. argus holds significant commercial value due to its high meat content, fewer intermuscular bones, and excellent meat quality compared to many freshwater fish species 3,4 .In 2020, China's annual production of C. argus surpassed 500,000 tons, resulting in over one billion US dollars in direct economic benefits.Additionally, it has provided employment and economic opportunities for hundreds of thousands of farmers 5 .Building upon these achievements, efforts have been made to breed superior varieties of C. argus.For instance, the growth rate of YY super-male snakeheads, a breed developed through sex reversal and molecular sexing, has been enhanced by 17.3% 6 .Furthermore, hybrid snakeheads obtained by crossbreeding Channa maculata (female parent) and C. argus (male parent) have been explored 7 .On www.nature.com/scientificreports/ the other hand, one notable drawback of wild snakeheads and hybrid snakeheads is their resemblance to snakes, which might deter some of the customers.Therefore, the development of new snakehead varieties with altered physical appearances holds great potential for driving advancements in related industries.
Albinism / leucism are common phenomena observed in C. argus within the Jialing River system and Tuojiang River system in Sichuan Province, China.It serves as a significant method for eliminating the serpent-like appearance, wherein the synthesis of pigments within the organism's body is obstructed, consequently leading to the manifestation of a white, golden-white, or light-golden exterior form 8 .The emergence of the yellowalbino variety may be attributed to the potential involvement of genes associated with pathways such as MAPK, WNT, and calcium signaling, which potentially increase melanogenesis elements and are likely stimulated by fibroblast-derived melanogenic factors 9 .On the other hand, the production of white varieties may be due to the fact that melanin synthesis, WNT, and MAPK signaling pathways are associated with skin depigmentation in C. argus, and the low expression of pigment-related genes contributes to the manifestation of whitening characteristics 10 .Recently, a new white (leucism) variety of C. argus, referred to as the "Chinese White Jade Dragon [variety registration number: GS-01-005-2022]" has gained popularity.This variety exhibits a snow-white body surface and yellow-gold fins, symbolizing wealth and prosperity, which has gradually entered the high-end food market (Fig. 1A) 11 .The white variety is sold at a price three times higher than the wild variety, providing greater economic benefits to farmers.Therefore, the development of white variety culture not only contributes to the conservation of C. argus diversity but also introduces novel and economically viable opportunities into traditional snakehead culture.
Building upon the observed morphological changes, the next stage in molecular breeding is to further extend the foundational traits of stable varieties, even amidst this process.Leucism, which gives rise to distinctive varieties, is a common occurrence in fish genetic variation 10 .Consequently, the selection and breeding of superior traits in white C. argus assume paramount importance.Notably, Zhou et al. 12 discovered that the white variety exhibits a higher N-3 to N-6 polyunsaturated fatty acids, including EPA+DHA, compared to the wild variety.Additionally, Wang et al. 13 demonstrated that the white variety possesses higher protein content and lower fat content in its body composition.These findings provide valuable breeding implications for white C. argus.However, the development disparities and molecular mechanisms underlying selective breeding to enhance the growth performance of the white variety remain unclear.Thus, it is crucial to investigate the potential influence of leucism on organ development and elucidate the underlying molecular mechanisms.The sequencing and gradual refinement of the C. argus genome offer a more reliable reference for investigating these molecular mechanisms 14 .Currently, transcriptomic sequencing has been conducted on a substantial number of relevant samples 15 , encompassing diverse areas such as infection 16 , toxicology 17 , and genetic development 18 .Moreover, the construction of a genetic linkage map has enabled the creation of a comprehensive sex-averaged map and sex-specific genetic maps for the C. argus 19,20 .In contrast, when it comes to the leucism variety of the C. argus, transcriptomic investigations have predominantly focused on unraveling the mechanisms underlying skin depigmentation 10 .

Comparative analysis of organ development and variation in white and wild varieties
Then, we have conducted comprehensive measurements and histological analyses to compare the organ indexes, coloration, and tissue structures of various organs such as the liver, spleen, kidney, heart, intestine, and gills in order to assess their developmental differences.Remarkably, notable variations were observed in two varieties in the indexes of liver, spleen, kidney, and intestinal at 150 dpf.Specifically, we found that the proportions of liver and intestinal indexes were significantly increased in the white variety (Fig. 2A,G), while the proportions of spleen and kidney indexes were significantly decreased compared to the wild variety (Fig. 2D, S1A).Furthermore, our organ color analysis revealed a significant paleness of the liver (see Supplementary Fig. S1E online), whereas no significant change was observed in the color of the spleen and kidney (see Supplementary Fig. S1B,F online).Histological studies of the liver showed normal morphology in both varieties at 120 dpf, with moderate granular degeneration observed at 150 dpf, but no significant difference was observed among different varieties (Fig. 2B,C).However, the area of the white pulp in the white variety was slightly decreased compared to the wild variety, particularly noticeable at 120 dpf, although the difference was not statistically significant (Fig. 2E,F).In contrast, the length of intestinal villus in the white variety was significantly higher than that of the wild variety at 120 dpf (Fig. 2H,I).Based on these findings, it appears that the white variety exhibits a bigger digestive system, which may contribute to its higher muscle index.Additionally, our observations implied that the white variety displays relatively weaker immune capacity.This was also suggested by renal histology, where the renal interstitial ratio of the white variety was lower than that of the wild variety (see Supplementary Fig. S1C,D online), indicating a relatively low hematopoietic immune function.Furthermore, no significant difference was observed in gills and heart indexes (see Supplementary Fig. S1G,H online), indicating similar respiration and pumping capacity in both varieties.

Microbiota composition and developmental differences in two C. argus varieties
The host provides an opportunity to bacteria for a symbiotic relationship 21,22 , which are involved in regulating internal satiety signals, digestion and absorption, neural pathways, immune pathways, and cytokines 23 .Thus, the magnitude of organism growth is hypothesized to be influenced by a combination of exogenous and endogenous genes, with the exogenous genetic elements potentially being regulated by the symbiotic bacterial populations residing within the host organism.In order to investigate this phenomenon, we extracted DNA from the contents of the mid-intestine to hindgut of two varieties of C. argus, and compared their intestinal microbiota composition using 16S-seq.A total of 110,572,855 valid sequences, with an average length of 426 bp, were obtained, as shown in Table 1.Annotation analysis identified 327 species (similarity: < 97%) across all samples, belonging to 216 genera and 16 phyla.Among these, 215 species were common to both varieties, while 58 and 54 species were exclusively annotated in the white and wild C. argus, respectively (Fig. 3A).Notably, samples from both varieties did not exhibit distinct clustering according to the PLS-DA analysis (Fig. 3B).Additionally, the diversity test revealed no significant difference in community diversity (Fig. 3C).The predominant bacteria found in the intestinal tracts of C. argus included Actinobacteriota, Bacteroidota, Proteobacteria, Spirochaetota, and Firmicutes (Fig. 3D), with no significant differences observed between the two varieties (Fig. 3E).These findings suggest that, within the same aquaculture environment, the white variety of C. argus did not exhibit distinct bacterial selection preferences compared to the wild variety.Furthermore, this indirectly indicates the negligible impact of the exogenous gene set, composed of symbiotic microbial communities, on the developmental differences between the two varieties.However, phenotypic analysis indicated that the white variety demonstrated a higher aerobic capacity compared to the wild variety, while this distinction did not exhibit statistically significant differences (P > 0.05) (Fig. 3F).Analysis of the larger species distribution within the two groups revealed that both varieties carried a high proportion of Aeromonas veronii, while variations were observed between the two varieties in terms Vol:.( 1234567890 www.nature.com/scientificreports/ of Plesiomonas shigelloides, family Mycoplasmataceae, Pseudomonas parafulva, and genus Shewanella (Fig. 3G).Despite these discernible variations, statistical analysis revealed a lack of significant disparities (P > 0.05).www.nature.com/scientificreports/

Unraveling the transcriptomic profile of selected organs in two varieties of C. argus
In order to explore the genetic differences underlying organ development induced by the host, we extracted mRNA from the livers and spleens of two varieties to construct cDNA libraries.The Illumina Novaseq 6000 platform was utilized for the sequencing process.Following the completion of data generation and quality control protocols, the Clean Data of all samples exceeded 6.16 Gb, with a Q30 base percentage of over 94.31%.Subsequently, the clean reads of the samples were aligned to the reference genome GCA_004786185.1 using RSEM (utilizing Bowtie 2 with a parameter mismatch of 0) for mapping analysis.The mapping rate of each sample was 90.29% ± 5.76%, which met the requirements for RNA-seq analysis utilizing a reference genome (Table 2).Data analysis was performed using Trinity, and annotation was conducted using Clusters of Orthologous Groups (COG), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of orthologous groups for eukaryotic complete genomes (KOG), Pfam, and Swiss-prot databases, resulting in the identification of 23,966 genes, including 17,048 known genes and 6,918 novel genes.Firstly, disregarding the specific varieties, we employed the DESeq2 algorithm with a screening threshold of |log2FC|> = 1 and P adjust < 0.05 to carry out differential gene analysis between two organs, aiming to obtain the transcriptome profiles related to organ development in C. argus.Consequently, we identified a total of 6787 genes that were commonly expressed, whereas 5798 genes exhibited differential expression in the spleen and liver.Notably, among these differentially expressed genes, 807 genes displayed higher expression levels in the liver, while 4991 genes exhibited elevated expression levels in the spleen (Fig. 4A-C).The GO enrichment analysis indicated that the differentially expressed genes (DEGs) were involved in Cellular components, Biological processes, and Molecular functions, exhibiting minimal disparity between the two organs (Fig. 4D).According to KEGG results, highly expressed genes in the liver were predominantly associated with Metabolism and Gene information processing, whereas highly expressed genes in the spleen were mainly linked to Human diseases, Environmental information processing, and pathways related to immunity and the excretory system (Fig. 4E).

Identification of DEGs in the liver and spleen in two C. argus varieties
Then, taking into account the expression differences between the two varieties in the respective organs, we performed separate comparative analyses of gene expression in the liver and spleen using the DESeq2 algorithm.www.nature.com/scientificreports/Our investigation unveiled a substantial number of expressed genes, with over 7000 genes in the liver and more than 10,000 genes in the spleen (Fig. 5A).Interestingly, a total of 6224 genes were found to be expressed in two organs examined (Fig. 5A).Despite the similarities observed in the overall expression patterns between the liver and spleen of both wild and white C. argus varieties (Fig. 5B), we identified 76 and 35 DEGs in the liver and spleen, respectively (Fig. 5C, see Supplementary Table 1 online).Notably, we found that the genes pcdhf 4, nlrc3 card 15-like, and a hypothetical protein gene containing a DNA/RNA polymerases domain (pol-like) were found to be differentially expressed both in the liver and spleen transcriptomes (Fig. 5D).Interestingly, nlrc3 card 15-like exhibited down-regulation, pcdhf 4 and pol-like showed up-regulation in white variety compared to the wild variety.

argus varieties
Furthermore, our analysis of DEGs allowed us to identify significant pathways enriched in the liver and spleen.
In the liver, we observed notable enrichment in pathways associated with Viral protein interaction with cytokine and cytokine receptor (VPIWC&CR), Estrogen signaling (ESP), Protein processing in the endoplasmic reticulum (PPIER), TNF signaling (TNFSP), and Cytokine-cytokine receptor interaction (CCRI) (Fig. 6A).Conversely, in the spleen, the primary enriched pathway was found to be related to Arginine and proline metabolism (A&PM) (Fig. 6A).Additionally, through gene ontology (GO) analysis, we discovered specific pathways concentrated in Anatomical structure development in the liver and Phosphorus metabolic process in the spleen (Fig. 6B, see Supplementary Figs.S2 and S3 online).Lastly, we conducted a thorough analysis of the transcriptome data, which led to the identification of numerous single nucleotide polymorphisms (SNP/InDel) differences predominantly located on chromosome CM015724.1 in the white variety (Fig. 6C).Moreover, we observed significant alternative splicing events in both the liver (226) and spleen (447) (Fig. 6D).

Discussion
Despite the observed pigmentation alterations associated with leucism in snakeheads, the precise molecular mechanisms underlying the enhanced growth performance of the white variety remain poorly understood.In this study, we sought to elucidate the biological factors contributing to this phenomenon.Our findings reveal that the white variety exhibits a relative higher growth rate, increased muscle index, and improved digestive area compared to the wild variety, all while displaying a relatively smaller immune tissues under identical feeding conditions.To gain further insights, we constructed a comprehensive transcriptional map of the liver and spleen of C. argus and conducted a thorough characterization of the gene expression profiles within these organs.
Through transcriptome analysis, we discovered numerous SNP/InDel events, predominantly located on chromosome CM015724.1.Furthermore, our annotation of liver and spleen genes unveiled 76 and 35 genes, respectively, displaying significant enrichment in pathways such as "VPIWC&CR", "ESP", "PPIER", "TNFSP", "CCRI", and "A&PM".These findings present potential molecular markers that can be utilized for the selection of the white variety.By extending our understanding of leucism beyond its superficial manifestations, this study highlights the remarkable traits associated with this phenotype and lays the groundwork for future molecular breeding programs aimed at further enhancing this variety.Organs serve as fundamental entities governing animal life processes and act as the material foundation for their physiological functions.The Organ index, to some extent, provides insight into the organ workload within the organism 26 .Our research findings indicate noticeable variations in the liver and intestinal indexes, a reduction in the spleen and kidney indexes, while no significant disparity in gill and heart.These observations imply that the white variety has undergone specific modifications in its digestive and immune systems, while its respiratory and circulatory capacities remain unaffected.Of particular interest, the liver and intestinal indexes of the white variety exhibited a remarkable increase of 8.5%, with no discernible histological distinctions.This observation implies that, at an equivalent cellular functional level, the white variety possesses a relatively higher proportion of functional areas for digestion and detoxification during its early development, which could accelerate the growth rate for aquaculture.Moreover, we detected higher expression levels of "ESP" which may potentially contribute to enhanced cell growth, transcription, apoptosis inhibition, and pathogen interaction 27 .These factors significantly impact individual development.Additionally, the muscle index displayed a noteworthy 10.5% increment, reflecting an enhancement in developmental capability, and suggesting that the white variety possibly possesses improved physical agility.Importantly, this translates to substantial benefits for aquaculturists, as it yields more profitable outcomes, making it a cost-effective commodity for consumers.
According to the results, the host possesses the ability to influence the selection of specific bacterial communities through immunoglobulin (Ig) A or IgM 21,22 .Based on the findings of this study, under the same rearing environment, no significant structural differences were observed between the microbial communities of the white and the wild varieties.This suggests that their selection ability remains unaffected by leucism.Considering the crucial role of intestinal microbiota in host growth and immune function, the unaltered colonization of these microbiota also largely eliminates potential impacts on the organ development of white variety.This helps to better explore potential genetic difference in the host itself.Nevertheless, at the genus level, the white variety exhibited higher levels of P. shigelloides and P. parafulva in its intestinal flora, which are opportunistic

Figure 1 .
Figure 1.The comparison regarding the growth characteristics of white and wild C. argus.(A) The appearance of the two varieties, wild and white C. argus, was depicted.(B), (C) The variations in body weight and body length, respectively, between the two varieties at 120 dpf and 150 dpf (ANOVA, n = 10).(D) The histology of skeletal muscle at 150 dpf, specifically highlighting the perimysium, as indicated by the arrows, using H&E staining at a magnification of × 40.(E) The muscle index (muscle weight/body weight × 100%) between two varieties (ANOVA, n = 10).Notably, the asterisk (*) denotes a statistically significant difference (P < 0.05) observed between the two varieties.

Figure 2 .
Figure 2. The analysis of organ indices and histological measurements in white and wild C. argus.(A), (D), & (G) The liver index, spleen index, and intestine index (organ weight/body weight × 100%) were examined for both white and wild C. argus, respectively (ANOVA, n = 10).(B), (E), & (H) The histology of liver (× 400), spleen (× 100), and intestine (× 40) was assessed through H&E staining in white and wild C. argus, respectively (n = 10).(C) The area of hepatic cells in the liver was measured and compared between the two varieties at 120 dpf and 150 dpf (ANOVA, n = 10).The average area of each liver cell in an individual is determined by dividing the area of liver tissue in the slice (excluding tissues like the pancreas and blood vessels) by the number of cell nuclei.(F)The ratio of red pulp to white pulp in the spleen was determined for both varieties at 120 dpf and 150 dpf (ANOVA, n = 10).The relevant ratio was obtained by dividing the area of the red pulp by the area of the white pulp.(I) The length of intestinal villus (IV) and muscular layer & serous membrane (MLSM) in the intestine was determined for both varieties at 120 dpf and 150 dpf (ANOVA, n = 10).All evaluations were performed using Image Processing and Analysis in Java (https:// imagej.net/ ij/), with the calculation rules for the corresponding area or length documented in the figure.Statistical significance was indicated by *, representing a significant difference (P < 0.05), or **, representing a highly significant difference (P < 0.01), between the two varieties.

Figure 3 .
Figure 3.The comparative analysis of the intestinal microbial community between two varieties.(A) Venn diagram illustrating the species overlap between the two varieties.(B) Partial Least Squares Discriminant Analysis (PLS-DA) plot depicting the distribution of samples from the two varieties.(C) Results of the alpha diversity test includes Shannon index, ACE index, Simpson index, and Chao index, assessing the diversity within each variety (t-tests, n = 3).(D) Phylogenetic tree displaying the bacterial species present in variety C. argus.(E) Relative abundance of different phyla in the two varieties (t-tests, n = 3).(F) Bugbase phenotype annotation, providing information on the phenotypic characteristics of the microbial species (t-tests, n = 3).(G) Distribution of the major bacterial taxa across different intestinal flora (t-tests, n = 3).

Figure 4 . 3 .
Figure 4. Comparative analysis of the liver and spleen transcriptomes identifies differentially expressed genes.(A) Venn diagram illustrating the overlap between the liver and spleen.(B) Scatter plot displaying the expression differences between the two organs.(C) Heat map depicting the differential expression in the liver and spleen.(D) Gene Ontology (GO) annotation of highly expressed genes in the spleen and liver24 .(E) Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation of highly expressed genes in the spleen and liver25 .n = 3.

Figure 5 .
Figure 5. Comparative analysis identified gene expression profiles in the liver and spleen of different varieties.(A) Upset diagram illustrating the distribution of gene expression patterns in the liver and spleen between different varieties.(B) Principal components analysis (PCA) plot showing the clustering of samples based on gene expression profiles in the liver and spleen.(C) Scatter plot depicting the differential expression of genes between the liver and spleen in different varieties.(D) Heat map representing the differential expression of genes in the liver and spleen between different varieties (Deseq2 & BH method).Genes marked with red indicate their presence in both the liver and spleen.n = 3.

Figure 6 .
Figure 6.Gene function annotation and structural variability analysis of liver and spleen in different varieties.(A) KEGG enrichment analysis of differentially expressed genes (DEGs) in liver and spleen 25 .(B) Gene Ontology (GO) annotation of DEGs in liver and spleen 24 .(C) SNP/InDel analysis comparing different varieties.(D) Analysis of alternative splicing in liver and spleen between varieties.

Table 2 .
Total quality of RNA samples.